Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the dehydration of carbonic acid into CO2 plus water, facilitates H+ secretion in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA IV on H+ secretion, we measured net HCO3- transport in perfused OMCDi segments and examined the effect on transport of two extracellular CA inhibitors, benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer, polyoxyethylene bis(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg, Chem, 24: 262-272, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net HCO3- flux were performed for each drug. Basal HCO3- absorptive flux was 12 pmol.min-1.mm-1 in control segments and significantly increased to 16 pmol.min-1.mm-1 in segments from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited HCO3- absorption by 50% were approximately 0.1 and approximately 5 microM, similar to the Ki for CA IV inhibition by these agents (0.2 and 4.0 microM, respectively; T. Maren, C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98-105, 1997). Adding exogenous CA to the inhibitor in the perfusate nearly restored basal HCO3- transport, suggesting that cytosolic CA II was not inhibited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited HCO3- absorption by 50% were 50 and 500 microM, respectively, > 100 times the Ki for CA IV inhibition and for inhibition of HCO3- transport in control tubules. Thus, in the OMCDi, doses of extracellular CA inhibitors that inhibited approximately 50% of CA IV activity also comparably inhibited HCO3- transport, indicating that H+ secretion depends in part on the availability of luminal CA IV activity. Acidosis substantially decreased the sensitivity of HCO3- transport to CA inhibition.